Chia seed derived products and the process thereof

ABSTRACT

The present application relates generally to a process of manufacturing a plurality of products from chia seeds. The process comprises socking chia seeds in a large volume of water for a period of time followed by sonication at an elevated temperature to remove the soluble fiber, the mucilage of chia seeds using vacuum filtration. The oil component is extracted from mucilage removed chia seeds and the leftover is hydrolyzed in presence of an enzyme to afford a product of protein hydrolysates and a product of insoluble fiber of chia seeds. Both the process and the products are within the scope of this application.

CROSS-REFERENCE TO RELATED APPLICATION

The present U.S. patent application relates to and claims the prioritybenefit of U.S. Provisional Application Ser. No. 62/947,125, filed Dec.12, 2019, the contents of which are hereby incorporated by reference intheir entirety.

SEQUENCE LISTING STATEMENT

A computer-readable form (CRF) of the Sequence Listing is submitted withthis application. The file, entitled 68853-02_Seq_Listing_ST25_txt, isgenerated on Dec. 2, 2020. Applicant states that the content of thecomputer-readable form is the same and the information recorded incomputer readable form is identical to the written sequence listing.

TECHNICAL FIELD

The present application relates generally to a process of manufacturinga plurality of products from chia seeds, comprising oil, soluble fibers,insoluble fibers, protein hydrolysates, and series small biologicalactive peptides. Both the process and the products are within the scopeof this application.

BACKGROUND AND SUMMARY

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

Chia (Salvia hispanica) has gained popularity due to its highnutritional content. Unfortunately, mucilage surrounding the chia seed(CS) limits the technological utilization of the protein. This studyevaluated the bioactivity and functionality of CS protein hydrolysates(CSPH) produced by different treatments and a control (unhydrolyzed CSprotein). Ultrasonication was used to separate mucilage from CS (7.8%yield). Proteins in defatted-CS flour were enzymatically hydrolyzedusing conventional (enzymatic hydrolysis with alcalase) or sequential(enzymatic hydrolysis with alcalase+flavourzyme), and under water bathor microwave-assisted hydrolysis. CSPH from the sequential hydrolysiswith microwave treatment showed superior (p<0.05) in vitro antioxidantactivity. A positive correlation (p<0.05) was established betweenantioxidant assays and cellular antioxidant activity. The highest(p<0.05) cellular antioxidant activity was achieved by the sequential(94.76±1.96) and conventional (93.13±1.07) hydrolysis with microwave.Dipeptidyl peptidase-V inhibition (p<0.05) was higher for sequentialhydrolysis with water bath. Inhibition of angiotensin converting enzymeactivity increased (p<0.05) with hydrolysis for all treatments comparedto the control. Regarding functionality, sequential hydrolysis withmicrowave showed higher (p<0.05) solubility at lower pH (3 and 5), whileconventional hydrolysis with microwave was better at pH 7 and 9.Emulsification properties and foaming capacity were also higher inconventional hydrolysis with microwave, but conventional hydrolysis withwater bath was more stable for foaming properties only. Results showthat ultrasonication efficiently separated mucilage from chia seeds.Microwave and enzymatic hydrolysis can generate protein hydrolysateswith improved bioactivity and functionality.

DRAWINGS AND BRIEF DESCRIPTIONS

FIGS. 1A-1C show the diagram of mucilage separation (extraction) fromchia seeds using ultrasonication and vacuum-assisted filtration (FIG.1A); clean chia seeds after mucilage extraction using ultrasound andvacuum-filtration separation (FIG. 1B); chia seeds with residualmucilage using ultrasound and centrifugation (FIG. 1C).

FIGS. 2A-2C shows results for solubility (FIG. 2A), emulsifying activity(FIG. 2B) and foaming capacity (FIG. 2C) of chia seed proteinhydrolysates. Different letters (a-d) show significant differences(p<0.05) between treatments. Sample codes descriptions are provided inTable 1.

FIG. 3 shows molecular weight distribution of chia seed proteinhydrolysates. Lanes indicate: (Lang 1) molecular weight markers, (Lang2) Control: unhydrolyzed chia seed flour (Lang 3) A-WB: chia seedprotein hydrolyzed by alcalase enzyme using water bath heating method.(Lang 4) A-MW: chia seed protein hydrolyzed by alcalase enzyme usingmicrowave-assisted hydrolysis. (Lang 5) AF-WB: chia seed proteinsequentially hydrolyzed by alcalase and flavourzyme enzymes using waterbath heating method. (Lang 6) AF-MW: chia seed protein sequentiallyhydrolyzed by alcalase and flavourzyme enzymes using microwave-assistedhydrolysis.

FIG. 4 describes a general process for manufacturing a plurality ofproducts from chia seeds.

FIG. 5 depicts enzyme-inhibition activity and IC50 values of the <3 kDapeptide fraction towards collagenase, hyaluronidase, tyrosinase, andelastase. The inhibitory activity of the peptide fraction was assayed at1 mg/mL and the IC50 (peptide concentration (mg/mL) required for 50% ofenzyme inhibition) was calculated using three different peptideconcentrations (1, 1.5, and 2 mg/mL). Values shown are mean oftriplicate determinations. Different lowercase (a-d) and uppercase (A-C)letters indicate statistical differences among IC50 values and % ofenzyme inhibition, respectively

FIGS. 6A-6D depict Lineweaver-Burk plots of the inhibitory patterns ofthe <3 kDa chia seed peptide fraction on (FIG. 6A) elastase, (FIG. 6B)collagenase, (FIG. 6C) hyaluronidase, and (FIG. 6D) tyrosinase, where1/[V] and 1/[S] represent the reciprocal of velocity and substrate,respectively

FIG. 7 shows size-exclusion chromatogram profile obtained from the <3kDa peptide fraction of chia seed hydrolysates

FIG. 8 depicts predicted models of interactions between elastase andeach peptide sequence obtained from the F-II chia seed peptide fraction.In each box, the left image represents the best accuracy predicted modelfor elastase (shown in white/multicolor) and peptide interaction (shownin red). The right image represents the hydrogen bonding betweenelastase (pink) and the peptide sequence tested (blue). The amino acidsinvolved in hydrogen bonding between the elastase and peptide aredepicted in green color. The amino acid sequence of the peptide testedis shown at the top of each image

FIG. 9 shows frequency of elastase-peptide amino acid interaction sitesin all molecular docking predicted models of peptide sequences obtainedfrom the F-II chia seed peptide fraction. T3105he protein sequenceshowed for elastase was generated using only the amino acids thatparticipated in all protein—peptide predicted models based on Table 2

FIG. 10 shows a schematic representation of the inhibitory mechanism ofchia peptides towards elastase.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended.

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

In the present disclosure the term “about” can allow for a degree ofvariability in a value or range, for example, within 20%, within 10%,within 5%, or within 1% of a stated value or of a stated limit of arange.

In the present disclosure the term “substantially” can allow for adegree of variability in a value or range, for example, within 80%,within 90%, within 95%, or within 99% of a stated value or of a statedlimit of a range. Soy and soybean are used exchangeably herein.

In this document, the terms “a,” “an,” or “the” are used to include oneor more than one unless the context clearly dictates otherwise. The term“or” is used to refer to a nonexclusive “or” unless otherwise indicated.In addition, it is to be understood that the phraseology or terminologyemployed herein, and not otherwise defined, is for the purpose ofdescription only and not of limitation. Any use of section headings isintended to aid reading of the document and is not to be interpreted aslimiting. Further, information that is relevant to a section heading mayoccur within or outside of that particular section. Furthermore, allpublications, patents, and patent documents referred to in this documentare incorporated by reference herein in their entirety, as thoughindividually incorporated by reference. In the event of inconsistentusages between this document and those documents so incorporated byreference, the usage in the incorporated reference should be consideredsupplementary to that of this document; for irreconcilableinconsistencies, the usage in this document controls.

In some illustrative embodiments, this disclosure relates to a processfor manufacturing a plurality of products from chia seeds comprising thesteps of:

-   -   a. socking chia seeds in about 10 to 20 volumes of water for a        period of time;    -   b. sonicating soaked chia seeds using 75% power input for about        5 minutes at an elevated temperature to afford a mixture;    -   c. separating chia seeds from said mixture by vacuum filtration        to afford a solution, which is lyophilized to afford a mucilage        product of chia seeds, a soluble fiber product;    -   d. drying mucilage-removed chia seeds; and    -   e. extracting oil from dried chia seeds at an elevated        temperature to afford an oil product and a flour of defatted        chia seeds.

In some illustrative embodiments, this disclosure relates to a processfor manufacturing a plurality of products from chia seeds as disclosedherein, wherein said process further comprising a step of hydrolyzingthe flour of chia seeds in an aqueous solution in presence an enzyme ata pH of about 6˜8.

In some illustrative embodiments, this disclosure relates to a processfor manufacturing a plurality of products from chia seeds as disclosedherein, wherein said hydrolyzing step is a microwave-assistedhydrolyzing process.

In some illustrative embodiments, this disclosure relates to a processfor manufacturing a plurality of products from chia seeds as disclosedherein, wherein said enzyme is alcalase with optional flavourzyme.

In some illustrative embodiments, this disclosure relates to a processfor manufacturing a plurality of products from chia seeds as disclosedherein, wherein said hydrolyzing step affords a solid product and asolution product after separation, wherein said solid product is aninsoluble fiber product of chia seed and said solution product is aprotein hydrolysate of chia seeds.

In some illustrative embodiments, this disclosure relates to a processfor manufacturing a plurality of products from chia seeds as disclosedherein, wherein said protein hydrolysate of chia seeds is furtherresolved into a plurality of fractions comprising biologically activeproteins and peptides.

In some illustrative embodiments, this disclosure relates to a processfor manufacturing a plurality of products from chia seeds as disclosedherein, wherein said active peptides have a sequence of APHWYTN (SEQ IDNO: 1), DQNPRSF (SEQ ID NO: 2), GDAHWAY, (SEQ ID NO: 3), GDAHWTY, (SEQID NO: 4), GDAHWVY (SEQ ID NO: 5), GFEWITF (SEQ ID NO: 6), KKLKRVYV (SEQID NO: 7), GDAHW (SEQ ID NO: 8), a salt or a derivative thereof.

As used herein, a derivative is a compound that is derived from asimilar compound by a chemical reaction, such as one atom or group ofatoms is replaced with another atom or group of atoms. In thisdisclosure, a product of the C-terminal amidation, or N-terminalacylation of those peptides disclosed herein are considered as aderivative of those peptides.

In some illustrative embodiments, this disclosure relates to a processfor manufacturing a plurality of products from chia seeds as disclosedherein, wherein said oil product of chia seeds has a yield of about 30%weight of starting chia seeds.

In some illustrative embodiments, this disclosure relates to a processfor manufacturing a plurality of products from chia seeds as disclosedherein, wherein said elevated temperature ranges from about 30° C. toabout 70° C.

In some other illustrative embodiments, this disclosure relates to aproduct manufactured according to the process disclosed herein, whereinsaid product is an oil of chia seeds, a mucilage of chia seeds, asoluble fiber product, or a soluble protein hydrolysate.

In some other illustrative embodiments, this disclosure relates to aproduct manufactured according to the process disclosed herein, whereinsaid soluble protein hydrolysate comprises a peptide, a salt, aderivative or a fragment thereof, having a sequence of SEQ ID NOs: 1, 2,3, 4, 5, 6, 7, or 8.

Yet in some other illustrative embodiments, this disclosure relates to aplurality of products of chia seeds manufactured according to a processcomprising the steps of:

-   -   a. socking chia seeds in about 10-20 volumes of water for a        period of time;    -   b. sonicating soaked chia seeds using 75% power input for about        5 minutes at an elevated temperature to afford a mixture;    -   c. separating chia seeds from said mixture by vacuum filtration        to afford a solution, which is lyophilized to afford a mucilage        product of chia seeds;    -   d. drying mucilage-removed chia seeds;    -   e. extracting oil from dried chia seeds at an elevated        temperature to afford an oil product and a flour of defatted        chia seeds; and    -   f. hydrolyzing the flour of defatted chia seeds in an aqueous        solution in presence an enzyme.

In some other illustrative embodiments, this disclosure relates to aplurality of products of chia seeds manufactured according to a processdisclosed herein, wherein the step of hydrolyzing the flour of defattedchia seeds affords a solid product and a solution product afterseparation, wherein said solid product is an insoluble fiber product ofchia seed and said solution product is a protein hydrolysate of chiaseeds.

In some other illustrative embodiments, this disclosure relates to aplurality of products of chia seeds manufactured according to a processdisclosed herein, wherein said hydrolyzing step is a microwave-assistedhydrolyzing process.

In some other illustrative embodiments, this disclosure relates to aplurality of products of chia seeds manufactured according to a processdisclosed herein, wherein said enzyme is alcalase optionally togetherwith flavourzyme.

In some other illustrative embodiments, this disclosure relates to aplurality of products of chia seeds manufactured according to a processdisclosed herein, wherein said protein hydrolysate of chia seeds isfurther resolved into a plurality of fractions comprising activeproteins and peptides.

In some other illustrative embodiments, this disclosure relates to aplurality of products of chia seeds manufactured according to a processdisclosed herein, wherein said biologically active peptides have asequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.

In some other illustrative embodiments, this disclosure relates to aplurality of products of chia seeds manufactured according to a processdisclosed herein, wherein said soluble protein hydrolysate comprises apeptide, a salt, a derivative or a fragment thereof, having a sequenceof SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.

In some other illustrative embodiments, this disclosure relates to aplurality of products of chia seeds manufactured according to a processdisclosed herein, wherein said elevated temperature ranges from about30° C. to about 70° C.

In some other illustrative embodiments, this disclosure relates to aplurality of products of chia seeds manufactured according to a processdisclosed herein, wherein said oil product of chia seeds has a yield ofabout 30% weight of starting chia seeds.

The present invention will become more clear in combination with thefollowing exemplified embodiments.

The following non-limiting exemplary embodiments are included herein tofurther illustrate the invention. These exemplary embodiments are notintended and should not be interpreted to limit the scope of theinvention in any way. It is also to be understood that numerousvariations of these exemplary embodiments are contemplated herein.

Throughout the years, plants have been domesticated and cultivated toobtain novel potential ingredients and alternative sources of functionalfoods. One remarkable plant that has shown high potential is chia(Salvia hispanica). Chia is a biannually cultivated plant; it isconsidered a pseudo-cereal that produces purple and white flowers thateventually result in small oval shape seeds with sizes varying from 1 to2 mm (Mohd Ali et al., 2012). The seeds are divided into twosemi-hemispherical structures, which contain the endosperm, calledcotyledons.

Nutritional composition (wet basis) of chia seed consists of protein(15-25%), lipids (30-33%), carbohydrates (26-41%), dietary fiber(18-30%) and minerals (4-5%) (Segura-Campos, Ciau-Solis, Rosado-Rubio,Chel-Guerrero, & Betancur-Ancona, 2014). Its composition allows the seedto have remarkable attributes such as a high content of protein,unsaturated (ω-3) fatty acids, and dietary fiber (Segura-Campos et al.,2014). The high dietary fiber content of chia seeds can be observed whenthe seeds are soaked in water and a copious mucilaginous polysaccharidecoating forms around the seed. This polysaccharide is present inmicrostructures called collumnellas that surround the chia seed andallow the formation of this gel-like material that limits itsdigestibility and utilization (Munoz, Cobos, Diaz, & Aguilera, 2012).The implementation of technologies such as ultrasound processing can aidin the separation of this polysaccharide, while allowing for proteinextraction (Vilkhu, Mawson, Simons, & Bates, 2008). Ultrasound methodshave shown high extraction yields in a shorter amount of time, for theseparation of the polysaccharide from other plant matrices such aslingzhi mushrooms (Ganoderma lucidum) and mutamba seeds (Guazumaulmifolia Lam) (Kang et al., 2019; Pereira et al., 2019).

A rising level of chronic diseases throughout the years have led to thedevelopment of food-derived bioactive peptides that can help improvethese medical conditions. Some of the biological effects produced bythese peptides are antioxidant, anti-inflammatory, anti-thrombotic,anti-hypertensive and anti-diabetic. Some proteins, including chia seedproteins, exhibit high resistance to proteolysis, limiting theirapplicability to generate bioactive peptides. For this reason, differenthydrolysis treatments such as high-voltage, electrical (Mikhaylin,Boussetta, Vorobiev, & Bazinet, 2017) and microwave (Nguyen, Jones, Kim,San Martin-Gonzalez, & Liceaga, 2017) treatments have been proposed toincrease the protein's susceptibility to hydrolysis.

Studies have shown the applicability and attributes of chia seeds. Forexample, one study showed how plasma α-linolenic acid andeicosapentaenoic acid increased by 58% and 39%, respectively when chiaseed was supplemented (25 g/day) in the diet of overweight women (Niemanet al., 2012). Another study by da Silva Marineli, Lenquiste, Moraes,and Maróstica Jr (2015), induced rats to overweight and oxidative stressbefore evaluating the effect of a diet rich in chia seeds on theseconditions, showing how plasma and hepatic antioxidant capacity valuesincreased. Sandoval-Oliveros and Paredes-López (2013) successfullyincorporated chia seed into drinks to enhance the protein content.

The aim of this study was to improve the biological and functionalproperties of chia seed protein hydrolysates by using ultrasonication toremove the mucilage and microwave-assisted enzymatic hydrolysis togenerate bioactive and functional chia seed peptides.

Materials and Methods

Chia seeds were obtained from Healthworks® (Scottsdale, Ariz., USA).Alcalase® (protease from Bacillus licheniformis, EC 3.4.21.62) andFlavourzyme® (protease from Aspergillus oryzae, EC 232-752-2) werepurchased from Sigma Aldrich (St. Luis, Mo., USA). Human DipeptidylPeptidase IV (DPP-IV, ≥4500 units/μg protein) and substrate Gly-Prop-nitroanilide hydrochloride, Angiotensin Converting Enzyme (ACE) fromhuman and substrate Hippuryl-L-Histidyl-1-Leucine (HHL) were allpurchased from Sigma Aldrich (St. Louis, Mo., USA). Elastase enzyme(from porcine pancreas, Type IV), N-(methoxysuccinyl)-Ala-Ala-Pro-Valp-nitroanilide, Tyrosinase enzyme (from mushroom),3,4-dihydroxy-L-phenylalanine, hyaluronic acid sodium salt (from Roostercomb), Hyaluronidase enzyme (from Bovine testes, Type I-S), Collagenaseenzyme (from Clostridium histolyticum, Type IA), and N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala, were purchased from Sigma-Aldrich (St. Louis,Mo., USA). All chemicals used were reagent grade and generally obtainedby three leading companies VWR International (Radnor, Pa., USA), SigmaAldrich (St. Louis, Mo., USA) and Thermo Fisher Scientific (Waltham,Mass., USA).

Chia Seed Mucilage Extraction

To extract the CS mucilage (FIG. 1A), seeds were hydrated in distilledwater (1:20 ratio by weight) for 24 h, under refrigerated conditions.Preliminary studies helped develop an ultrasound treatment that offeredsuccessful mucilage separation. Hydrated seeds were pre-heated to 55±2°C., followed by sonication at a 75% power input using an ultrasonic celldisruptor (Sonifier® Branson S-150D Danbury, Conn., USA). Duringsonication, the temperature increased to 60±4° C. due to molecularfriction. This temperature was maintained constant using double walledbeaker connected to an immersion circulator control Lauda E100 waterbath (Lauda-Königshofen, Germany). Seed were separated usingvacuum-assisted filtration.

Mucilage-free CS were dried using a tray dryer (Excalibur Dehydrator3926TCDB, Sacramento, Calif.) held at 40° C. for 12 h. The weight of theseeds was measured to calculate mucilage extraction yield by weightdifference [Eq. (1)]. Ultrasound mucilage extraction was compared toconventional extraction methods using drying oven and freeze-dryingtechniques following the methodology proposed by Campos, Ruivo, da SilvaScapim, Madrona, and Bergamasco (2016) and Capitani, Ixtaina, Nolasco,and Tomas (2013), respectively.

$\begin{matrix}{{\% \mspace{14mu} {yield}} = {\frac{\begin{pmatrix}{{{Weight}\mspace{14mu} {of}\mspace{14mu} {dry}\mspace{14mu} {chia}\mspace{14mu} {seeds}} -} \\{{Weight}\mspace{14mu} {of}\mspace{14mu} {dry}\mspace{14mu} {chia}\mspace{14mu} {seeds}\mspace{14mu} {without}\mspace{14mu} {mucilage}}\end{pmatrix}}{{Weight}\mspace{14mu} {of}\mspace{14mu} {dry}\mspace{14mu} {chia}\mspace{14mu} {seeds}} \times 100}} & (1)\end{matrix}$

Chia Seed Oil Extraction

Dried, mucilage-free CS were defatted using a mechanical oil extractionmethod with a Beamnova Automatic Oil Press Machine (Commercial 304Stainless Steel Expeller, Guangzhou, China). Seeds were pressed using astainless-steel endless screw held at 37±2° C. The defatted chia seedwas referred to as chia flour. Percentage of oil extraction wascalculated by weight difference [Eq. (2)].

$\begin{matrix}{{{yield}\mspace{14mu} \%} = {100 \times \frac{\begin{pmatrix}{{{Weight}\mspace{14mu} {of}\mspace{14mu} {chia}\mspace{14mu} {seeds}} -} \\{{Weight}\mspace{14mu} {of}\mspace{14mu} {defatted}\mspace{14mu} {chia}\mspace{14mu} {seeds}\mspace{14mu} {flour}}\end{pmatrix}}{{Weight}\mspace{14mu} {of}\mspace{14mu} {chia}\mspace{14mu} {seeds}}}} & (2)\end{matrix}$

In addition, the crude protein content of the chia flour was determinedusing AOAC methods 984.13 (A-D) by A&L Greatlakes laboratories Facility(Fort Wayne, Ind., USA).

Chia seed protein hydrolysate (CSPH)

Chia flour was diluted in distilled water to obtain 22.5 mg protein/mLand homogenized using a Sorvall Omni Mixer homogenizer with amacro-attachment assembly (Norwalk, Conn., U.S.A). The pH was adjustedto 8.0 using 2 M NaOH, which is the optimal condition for alcalaseactivity. Proteins were enzymatically hydrolyzed using single enzymealcalase (A) or a sequential digestion with alcalase+flavourzyme (AF).Proteolysis occurred using conventional (WB) or microwave-assisted (MW)hydrolysis using a microwave accelerated reaction system (MDS,MARS-Xpress/230/60, CEM Corporation, USA). Treatments were denoted asconventional alcalase hydrolysis using a water bath (A-WB) and alcalasemicrowave-assisted hydrolysis (A-MW). Sequentially (AF) hydrolyzedtreatments were coded as AF-WB (water bath hydrolysis) and AF-MW(microwave-assisted hydrolysis). Finally, the control (C) wasnon-hydrolyzed CS protein. Samples A-WB and A-MW were hydrolyzed for 1 hwith 3% (w/w) Alcalase®. For sequential hydrolysis different times wereused, due to the high efficiency of microwave-assisted hydrolysis thetime was cut down by half to obtain similar degree of hydrolysis. AF-MW,the reaction was initiated with 2% (w/w) of Alcalase® for 45 minfollowed by addition of 2% (w/w) of Flavourzyme® for an additional 45min. For AF-WB, the reaction was developed using 2% (w/w) of Alcalase®for 90 min followed by 2% (w/w) of Flavourzyme® for another 90 min.Hydrolysis was terminated by heating to 95±3° C. for 15 min.

Determination of the Degree of Hydrolysis

The degree of hydrolysis was calculated following the methodology ofAdler-Nissen with slight modifications by Liceaga-Gesualdo and Li-Chan(1999), measuring spectrophotometrically the color formed by free aminogroups reacting with Trinitrobenzenesulforonic acid (TNBS). The degreeof hydrolysis (% DH) was defined as a percent ratio of the number ofpeptide bonds broken (h) to the total number of peptide bonds per unitweight (htot). The htot was calculated experimentally using the fullyhydrolyzed chia seed flour, obtaining a value of 9.33 meq/g. The % DHwas calculated using equation [Eq. (3)].

$\begin{matrix}{{\% \mspace{14mu} {Degree}\mspace{14mu} {of}\mspace{14mu} {hydrolysis}\mspace{14mu} ({DH})} = \frac{h}{h_{tot}}} & (3)\end{matrix}$

Amino Acid Analysis

Total amino acid composition of CSPH was analyzed by the methoddescribed by Hall, F. G., Jones, O. G., O'Haire, M. E., and Liceaga(2017) by UPLC Amino Acid Analysis Solution using the AccQ Tag UltraDerivatization kit with UV detection (Water Corporations, Milford,Mass., USA) by the Danforth Center's Proteomics and Mass SpectrometryFacility (St. Louis, Mo., USA).

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

CSPH and control samples were dissolved to 2 mg/mL inzwitterionic-chaotrophic buffer according to Hall, Johnson, and Liceaga(2018) (2D-gel extraction buffer; 50 mM Tris-HCl, pH 8.8, 10 mMethylenediaminetetraacetic acid (EDTA), 5 M urea, 2 M thiourea, 67 mMDithiothreitol (DTT) and stirred for 1 h at room temperature. An aliquot(50 μL) was added to 50 μL Laemmeli sample buffer to obtain a 1 mg/mLfinal concentration of protein. A sample (20 μL) of this solution wasloaded using 4-12% gradient gels (Bis-Tris, NuPAGE, ThermoScientific,Waltham, Mass.) and ran with MES SDS running buffer (NuPAGE,ThermoScientific, Waltham, Mass.) at 200 V for 45 min. The gel wasstained overnight using Coomassie R-250 and destained overnight using asolution of 40% (v/v) methanol and glacial acetic acid. The molecularweight distribution of hydrolyzed peptides was determined usingPrecision plus Protein™ Dual Xtra Prestained Protein Standards (Biorad,Hercules, Calif.).

Bioactive Properties of CSPH

2-Diphenyl-2-Picrylhydrazyl (DPPH) Radical Scavenging Activity

The scavenging activity of the CSPH was determined according to a methoddescribed by

Bersuder, Hole, and Smith (1998) with modifications by Hall et al.(2018). CSPH and control (100 μL) were placed in a 96-well microplate towhich 100 μL of (99.5%) ethanol and 25 μL of DPPH solution at aconcentration of 0.05% (DPPH/ethanol, w/v) was added. The solution wasincubated for 30 min at room temperature in dark conditions, and theabsorbance measured at 550 nm using a microplate photometer. Radicalreduction was expressed in mM TE/mg sample. Absorbance values werecorrected using a sample blank prepared using 25 μL ethanol instead ofDPPH solution.

2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radicalscavenging activity (ABTS). The ABTS radical scavenging activity wasdetermined according to Ketnawa and Liceaga, (2017) with somemodifications. A solution of 7 mM of ABTS was prepared in 2.45 mM ofpotassium persulphate and incubated at room temperature for 16 h. After16 h the ABTS stock solution was diluted with distilled water to obtainan absorbance at 734 nm of 0.700±0.02. A CSPH sample aliquot (20 μL) wasmixed with 980 μL ABTS solution and incubated in the darkness at 30° C.for 10 min, followed by absorbance reading at 734 nm. The ABTSscavenging activity was calculated by equation [Eq. (4)] and resultsexpressed as mM Trolox equivalent (TE)/mg sample.

$\begin{matrix}{{{ABTS}\mspace{14mu} {scavenging}\mspace{14mu} {activity}} = {\frac{\left( {{{Abs}\mspace{14mu} {of}\mspace{14mu} {control}} - {{Abs}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}} \right)}{\left( {{Absorbance}\mspace{14mu} {of}\mspace{14mu} {control}} \right)} \times 100}} & (4)\end{matrix}$

Metal Ion Chelating (MIC)

The MIC capacity was done following the procedure by Ketnawa andLiceaga, (2017) with modifications. In a 96-well microplate, 200 μL ofCSPH samples were mixed with 3.75 μL of 2 mM FeCl2 and 7.5 μL of 5 mMFerrozine solution. Sample were incubated in the dark for 10 min at roomtemperature, and the absorbance was read at 522 nm. The MIC capacity wasCalculated using the equation [Eq. (5)].

$\begin{matrix}{{{MIC}\mspace{14mu} {ability}} = {\left\lbrack \frac{\left( {{{Abs}\mspace{14mu} {control}} - {{Abs}\mspace{14mu} {sample}}} \right)}{\left( {{Abs}\mspace{14mu} {control}} \right)} \right\rbrack \times 100}} & (5)\end{matrix}$

Oxygen Radical Absorbance Capacity (ORAC)

ORAC was measured according to a modified methodology described by Ou,Hampsch-Woodill, and Prior (2001). CSPH were diluted to a proteinconcentration of 0.05 mg/mL in a 75 mM sodium phosphate buffer at a pHof 7.4. The experiment was carried out in a 96-well microplate, eachwell containing a total volume of 205 μL 150 μL of Fluorescein (10 nM)was pre-incubated with 25 μL of CSPH sample solution for 15 min at 37°C. in dark conditions. Then the reaction was initiated by adding 30 μLof 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and theFluorescence was measured using a spectrophotometer (Fluoroskan AscentFL Microplate Fluorometer and Luminometer, Thermo Fisher Scientific,Massachusetts, United States) every 2 min for a total time of 90 minusing an excitation wavelength of 485 nm and emission of wavelength 535nm. The results were expressed in μM of Trolox Equivalent by measuringthe fluorescein area decay through time (AUC). The values werecalculated using the equation [Eq. (6)].

$\begin{matrix}{{{AUC} = {1 + \frac{f_{2}}{f_{0}} + \frac{f_{4}}{f_{0}} + \frac{f_{6}}{f_{0}} + \frac{f_{8}}{f_{0}} + {\ldots \mspace{14mu} {f_{90}/{f_{0}.}}}}},} & (6)\end{matrix}$

wherein f₀ represents the area under the curve at time 0 and fn theabsorbance taken every minute.

Cellular Antioxidant Activity (CAA)

The CAA was evaluated following the methodology proposed by Malaypallyet al.

(2015) and Wan, Liu, Yu, Sun, and Li (2015). First, CSPH and controlwere solubilized in Dulbecco's modified Eagle's medium (DMEM)/highmodified phenol red free. Caco-2 cells (100 μL, density of 7.6×105cells/mL) were placed in a 96-well black microplate and incubated for aperiod of 36 h under 5% CO₂ at 37° C. After this, the growth medium wasremoved using needles, washed using 1×PBS (100 μL), and exposed for 1 hto 100 μL of DMEM (60 μM Dichloro-dihydro-fluorescein diacetateDCFH-DA). CSPH was then added to a final concentration of 5 mg/mL. Thesolution was removed from each well, followed by a final washed-out with1×PBS. The cells were exposed to an oxidizing environment by pipetting100 μL of 500 μM AAPH into each well. Emission by the samples wasmeasured for every 5 min for 1 h using a fluoresce reader Spectra MaxGemini EM spectrofluorometer (Molecular Devices, Sunnyvale, Calif.) withan excitation wavelength of 485 nm and an emission wavelength of 538 nm.A sample blank, positive and negative control were required to calculatethe cellular antioxidant activity. Sample blanks contained DMEM and(DCFH-DA) without AAPH, negative control wells were prepared incubatingcells with DCFH-DA and AAPH and the positive control wells wereincubated with cells treated with 1-ascorbic acid (50 μM), DCFH-DA andAAPH. Finally, the cellular antioxidant activity was measured withequation [Eq. (7)], were the blank was subtracted from the samplereadings. The fluorescence emission against time data were plotted andused to, calculate the area under the curve in CAA values (%) (Wolfe &Liu, 2007)

$\begin{matrix}{{{CAA}\mspace{14mu} {unit}} = {100 - {\left( \frac{\int{SA}}{\int{CA}} \right) \times 100}}} & (7)\end{matrix}$

wherein ∫SA refers to the integral of the sample fluorescence vs. timeand ∫CA refers to the integral from the control sample.

Dipeptidyl peptidase IV (DPP-IV) inhibitory activity

The DPP-IV inhibitory activity of CSPH was determined following themethod by Hall et al. (2018). CSPH samples were dissolved in 100 mMTris-HCl buffer (pH 8.0) to a final concentration of 1.25 mg/mL. Samplealiquots (25 μL) were pipetted and pre-incubated in a 96-well microplatewith 25 μL of substrate Gly-Pro p-nitroanilide hydrochloride (6 mM) at37° C. for 10 min. The colorimetric reaction was initiated by adding 50μL of human DPP-IV (4.5 unit/mL), followed by incubation at 37° C. for60 min. The reaction was stopped by adding 100 μL of 1 M sodium acetatebuffer (pH 4.0). Absorbance of released p-nitroanilide was measured at405 nm using a Multiskan™ FC Microplate Photometer (Waltham, Mass.,USA). Sample absorbance was corrected by subtracting blanks in whichDPP-IV was replaced with Tris-HCl buffer (100 mM, pH 8.0). The positivecontrol (no inhibitor) used the buffer instead of CSPH sample. Fornegative control (no DPPIV activity), the buffer was used instead ofDPP-IV solution. Percent DPP-IV inhibition was calculated using equation[Eq. (8)].

$\begin{matrix}{{{DPP}\mspace{14mu} {IV}\mspace{14mu} {inhibition}\mspace{14mu} \%} = {1 - \left\lbrack \frac{{{Abs}\mspace{14mu} {Sample}} - {{Abs}\mspace{14mu} {Sample}\mspace{14mu} {blank}}}{{{Abs}\mspace{14mu} {positive}\mspace{14mu} {control}} - {{Abs}\mspace{14mu} {negative}\mspace{14mu} {control}}} \right\rbrack}} & (8)\end{matrix}$

Angiotensin Converting Enzyme (ACE) Inhibitory Activity

ACE-inhibitory activity was measured according to Hall et al. (2018).CSPH and control samples were dissolved in sodium phosphate buffer (100mM, pH 8.3) with NaCl (300 mM). Aliquots (25 μL) of CSPH solutions wereadded to 25 μL of the substrate hippuryl-L-histidyl-1-leucine (HHL) andincubated at 37° C. for 4 min. Then, aliquots (80 μl) of human-ACE (50mU) were added to initiate the reaction, followed by incubation at 37°C. in a water bath with constant stirring for 2 h. Reaction wasterminated by adding 50 μl of 1 M HCl; then the solution was filteredusing a 0.22 μm nylon filter. A control reaction was performed using 25μl of buffer instead of the inhibitor (CSPH). Hippuric acid (HA) wasquantified using high-pressure liquid chromatography (HPLC) (Model 600E,Waters Corporation, Milford Mass.) with a C18 analytical column (YMCPack ODS AM 12505-2546WT, YMC America, Inc., Allentown, Pa., USA).Percentage inhibition was calculated using equation [Eq. (9)]

$\begin{matrix}{{{{ACE}\mspace{14mu} {Inhibition}\mspace{14mu} \%} = {\left\lbrack {1 - \frac{A_{inhibitor}}{A_{control}}} \right\rbrack \times 100}},} & (9)\end{matrix}$

wherein, Ainhibitor and Acontrol represent the relative areas (A) withand without inhibitor of the HA peaks. The half maximal inhibitoryconcentration (IC50) determined the potency of the samples towards ACEinhibitory activity. IC50 was expressed in mg of protein per mL. IC50calculations were determined at four different CSPH concentrations(0.05, 0.5, 1, and 5 mg/mL) in triplicate.

Functional Properties of CSPH

Solubility. Protein solubility was measured following the methoddescribed by Chobert, Bertrand-Harb, and Nicolas (1988) and modified byHall, Jones, O'Haire, and Liceaga (2017). CSPH were diluted to 1 mg/mLin 15 mL buffers with pH 3.0 (0.1 M sodium Acetate), 5.0 (0.1 M sodiumAcetate), 7.0 (7.4 mM Phosphate) and 9.0 (0.1 M Glycine-sodiumhydroxide), respectively. The solutions were stirred for 30 min at roomtemperature, followed by centrifugation at 12,150×g (25° C.) for 5 min.The protein content in the supernatant was determined using thebicinchoninic acid (BCA) protein assay method with bovine serum albuminas standard. Protein solubility was calculated by the ratio of proteinin the supernatant to the protein content in the sample [Eq. (10)].

$\begin{matrix}{{{Solubility}\mspace{14mu} \%} = {\frac{{Protein}\mspace{14mu} {content}\mspace{14mu} {in}\mspace{14mu} {supernatant}}{{Total}\mspace{14mu} {protein}\mspace{14mu} {content}\mspace{14mu} {in}\mspace{14mu} {sample}} \times 100}} & (10)\end{matrix}$

Emulsion and Foaming Capacity

Emulsifying activity index (EAI) and Emulsion stability index (ESI) weremeasured spectro turbidimetrically following the procedure of Pearce andKinsella (1978) and some modifications by Liceaga-Gesualdo and Li-Chan(1999). Foaming capacity (FC) and foam stability (FS) were determinedusing the method proposed by Waniska and Minsella (1979) withmodifications of Pacheco-Aguilar, Mazorra-Manzano, and Ramirez-Suarez(2008).

Preparation of Chia Seed Protein Hydrolysates and their PeptideFractions

Hydrolysates from chia seed protein were obtained according toUrbizo-Reyes et al. (2019). First, chia seed mucilage was extractedusing a combined approach consisting of an ultrasound treatment andvacuum-assisted filtration. Subsequently, defatted chia seed meal washydrolyzed using sequential enzymatic (alcalase followed by flavourzyme,AF) microwave-assisted hydrolysis (MW). Subsequently, the hydrolysatewas fractionated by ultrafiltration using a <3 kDa cutoff membrane.Protein concentration was determined by the bicinchoninic acid (BCA)protein assay kit according to the manufacturer's instructions (PierceBiotechnology Inc., Rockford, Ill., USA), using bovine serum albumin asstandard. The protein concentration of each sample was adjusted to 1mg/mL using Tris-HCl buffer (100 mM, pH 8) for elastase,phosphate-buffered saline solution (PBS, 0.5 mM, pH 7.2) for tyrosinase,tricine buffer (50 mM, pH 7.5) for collagenase, and acetate buffer (50mM, pH 4.5) for the hyaluronidase inhibition assays. Additionally, forthe size exclusion chromatography analysis, the sample was dissolvedusing sodium phosphate (3.3 mM of Na₂HPO₄.7H₂O, 1.7 mM of NaH₂PO₄.H₂O)with 0.14 M sodium chloride at pH 7. All samples were immediately storedin the dark at 4° C. until used.

Evaluation of In Vitro Anti-Aging Bioactive Properties

Elastase Inhibition Assay of <3 kDa Fraction

The elastase inhibition was evaluated according to Azmi et al. (2014)with some modifications. Briefly, 100 μL of either test sample or buffer(control) were combined with 50 μL of substrate 10 mM/ofN-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (10 mM), andincubated for 15 min at 37° C. The solutions were mixed with 50 μL ofpre-incubated (5 min, 37° C.) elastase (50 mU), and the reaction wascarried out for 15 min. Finally, the absorbance of the reaction wasrecorded at 405 nm. The percentage of inhibition was calculated withequation [11], where OD_(control) and OD_(sample) represent the opticaldensity of the control and samples, respectively.

$\begin{matrix}{(\%)\mspace{14mu} {Inhibiiton}{= {\frac{{OD_{control}} - {OD_{sample}}}{OD_{control}} \times 100}}} & \lbrack 11\rbrack\end{matrix}$

Tyrosinase Inhibition Assay

Tyrosinase inhibition was evaluated according to Hong et al. (2019) withsome modifications. Briefly, 100 μL of either test samples or buffer(control) were combined with 50 μL of substrate3,4-dihydroxy-L-phenylalanine (10 mM), and incubated for 15 min at 30°C. The solutions were mixed with 50 μL of pre-incubated (5 min, 30° C.)tyrosinase (150 U), and the reaction was carried out for 15 min.Finally, the absorbance of the reaction was recorded at 450 nm. Thepercentage of inhibition was determined using equation [11].

Collagenase Inhibition Assay

Collagenase inhibition was evaluated according to the method by Hong etal. (2019) with some modifications. Briefly, 100 μL of either testsamples or buffer (control) were combined with 50 μL of substrateN-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (1 mM), and incubated for 15min at 30° C. The solutions were mixed with 50 μL of pre-incubated (5min, 30° C.) collagenase (100 mU), and the reaction was carried out for15 min. Finally, the absorbance of the reaction was recorded at 340 nm.The percentage of inhibition was calculated using equation [11].

Hyaluronidase Inhibition Assay

The hyaluronidase inhibition was evaluated according to Facino et al.(1995) with some modifications. Briefly, 100 μL of either test samplesor buffer (control) were combined with 50 μL of substrate hyaluronicacid sodium salt, and incubated for 15 min at 37° C. The solutions weremixed with 50 μL of pre-incubated (5 min, 37° C.) hyaluronidase (1mg/mL), and the reaction was carried out for 15 min. Absorbance wasrecorded at 550 nm. The percentage of inhibition was calculated usingequation [11].

For all the inhibitory activity assays, in addition to the percent ofenzyme inhibition (at 1 mg/mL), the peptide concentration (mg/mL)required for 50% enzyme inhibition (IC50) was calculated using threedifferent peptide concentrations (1, 1.5, and 2 mg/mL).

Mode of Enzyme Inhibition

The type of enzyme inhibition was determined according toLineweaver-Burk graphs of reciprocal product concentration absorbanceversus the reciprocal of substrate concentrations as described by Halland Liceaga (2020) with Km and Vmax were determined according to theLineweaver-Burk plots. Various substrate concentrations (0.5, 0.25, and0.125 mM) were incubated in the presence/absence of inhibitorconcentrations (0, 1, 1.5, and 2 mg/mL). Enzyme inhibition (%) weremeasured as described above. Reaction velocities (ΔAbs/min) weredetermined from the formation of product over time.

Identification and Characterization of Peptides in the Most Active <3kDa Peptide Fraction

The molecular weight distribution of the <3 kDa peptide fraction wasfirst determined using size exclusion chromatography (SEC) in a Waters2695 HPLC system equipped with a UV detection of 254 nm on a columnSuperdex peptide 10/300 GL (GE Healthcare, 17-5176-01, column 30 cm×10mm, 13 μm). Injection volume was 100 uL, and SEC eluent was sodiumphosphate (3.3 mM of Na₂HPO4.7H2O, 1.7 mM of NaH2PO4.H2O, with 0.14 Msodium chloride at pH 7) at flow rate of 1 mL/min. Signal was monitoredat 254 nm, and fractions were pooled and lyophilized immediately.Fractions (F-I, II, III, IV and V) were collected at different retentiontimes and the spectra normalized by their maximum peak height. Finally,before further analyses the samples were desalted using C18 desaltingtips (Thermo Scientific™ Pierce™) following manufacturer instructions.All the fractions collected were assayed for aging-related enzymeinhibitory activities according to the methods described above. The SECfraction with the highest inhibiting aging-related enzymes was furtherselected for peptide sequence identification by LC-MS/MS technique inthe Proteomics Core facility at the Indiana University School ofMedicine (Indianapolis, Ind., USA).

In Silico Analysis of F-II Peptides with Elastase

For this analysis, the peptides identified in the previous step and theenzyme elastase were used. The molecular protein-peptide interaction wasperformed using the CABS-dock web server(http://www.biocomp.chem.uw.edu.pl/CABSdock/) since it provides aninterface for modeling protein-peptide interactions using a highlyefficient protocol for the flexible docking of peptides to proteins(Kurcinski et al. 2015). Because the CABS-dock web server allows amaximum protein size of 500 amino acids for docking analysis, weselected elastase that has 240 amino acids, as the target enzyme. Inaddition, the chia seed peptides showed the greatest inhibition towardsthis enzyme. The 3D crystal structure of elastase from porcine pancreaswas downloaded from the Research Collaboratory for StructuralBioinformatics Protein Data Bank (RCSB PDB, https://www.rcsb.org/) withthe PDB ID: 1LVY. The analysis was performed with 50 cycles of MonteCarlo simulation. Of the final models (a set of 10 representativemodels), we select the top-ranked model 1 because is the most probabledocking trajectory and the best accuracy predicted model forelastase-peptide interaction. A Complementary analysis of the resultingstructures were done with the web-based 3D structure viewer “iCn3D”(www.ncbi.nlm.nih.gov) in order to observe the hydrogen bonding betweenelastase and the peptide tested.

Statistical Analysis

Results were reported as mean±standard deviation (SD) of triplicatedeterminations. A complete randomized design was used as a statisticalmodel with a Duncan separation of means p<0.05. The correlation analysisbetween antioxidant assays and cellular antioxidant activity wascalculated using a p<0.05. The statistical analysis was carried outusing the statistical software SAS 9.4 (Cary, N.C., USA). Thestatistical analysis of experimental data was made using ANOVA followedby Tukey's test. Differences were considered to be significant whenP<0.05. All analyses were performed using the NCSS software version 2007(NCSS Statistical software, Kaysville, Utah, USA). Each experiment wasrepeated three times, and all tests were run in triplicate for eachexperiment.

Results and Discussion

Mucilage Ultrasound Separation

In preliminary trials ultrasound treatment and vacuum-assistedfiltration increased mucilage separation the most, while other testedmethods involving other physical separation methods (centrifugation andmesh filtration) showed inefficient separation of mucilage (FIGS. 1B and1C). The mucilage ultrasound-extraction method was optimized using awave amplitude of 90 μm and 60° C. temperature, followed by separationusing vacuum-assisted filtration. The mucilage extraction yield washigher (p<0.05) when ultrasound was used (7.65±0.19%), compared tofreeze-drying the CS (4.21±0.29%), and oven drying CS (3.65±0.18%). Theyield values obtained for conventional extractions methods in this studylower than the ones reported by Campos et al. (2016) and Capitani et al.(2013). Chemat et al. (2017) showed how ultrasound developed high shearstresses in the proximity between liquid and solid materials, causingoil to separate from basil leaves, addressing the possibility of thisapplication in other food matrices such as chia seed mucilage.Ultrasound frequencies generated microjets in the chia seed surface,targeting structures called collumnellas that allowed for the physicalseparation of mucilage in a fast and efficient way. Mucilage extractioncan assist in its application as a biomaterial that can providestability to emulsions among other uses (Avila-De La Rosa,Alvarez-Ramirez, Vernon-Carter, Carrillo-Navas, & Perez-Alonso, 2015).We can conclude that the combination of ultrasound treatment followed byvacuum filtration increased the extraction yield of mucilage from chiaseeds.

Chia Seed Oil Extraction

The oil extraction yield obtained by cold press was 28.24±0.06 g ofoil/100 g of chia seeds (without mucilage). The oil content in chiaseeds varies between 30 and 33% (Sandoval-Oliveros & Paredes-López,2013). This pressing technology allows to remove about 86-94% of thetotal fat of the seed. The use of screw-press for oil extraction is agood alternative to reduce the use of hazardous solvents such asisopropanol and hexane in the extraction. Also, removal of oil fromseeds (in preliminary experiments) showed to increase the enzyme-proteininteraction, facilitating the hydrolysis.

TABLE 1 Hydrolysis conditions, degree of hydrolysis and bioactiveproperties of CSPH* Hydrolysis Degree of % DPPH ABTS ORAC time, (%hydrolysis MIC (μmol (μmol (μmol code Enzyme) (%) Inhibition TE/mg)TE/mg) TE/mg) A-WB 1 h 33.64 ± 1.44c 66.93 ± 0.57c 131.74 ± 17.33b 465.97 ± 10.46c  1225.49 ± 55.53bc 3% A-MW 1 h  37.04 ± 2.67bc 72.76 ±0.42b 166.61 ± 15.34a  435.30 ± 12.96d 1535.81 ± 99.16a 3% AF-WB 3 h46.81 ± 0.19a 74.11 ± 0.64b 171.31 ± 8.27a  489.09 ± 3.86b   1482.48 ±114.63 ab 2% AF-MW 1.5 h  40.68 ± 0.77 ab 76.85 ± 0.37a 178.02 ± 15.86a506.07 ± 4.50a 1122.71 ± 24.74c 2% Control 0  0.00 ± 0.00d 54.43 ± 1.03d22.99 ± 1.49c 100.57 ± 3.80e  816.33 ± 86.18d 0% DPPIV Cellularinhibition ACE inhibition antioxidant capacity capacity activity % % %per inhibition inhibition mg of per 2.5 mg IC₅₀ per mg of IC₅₀ codeprotein of protein (mg/mL) protein (mg/mL) A-WB  87.54 ± 3.31bc 37.05 ±0.99c 4.38 ± 0.49a 57.16 ± 1.93a 0.51 ± 0.12a A-MW  93.13 ± 1.07 ab40.56 ± 0.42c 3.59 ± 0.31b 57.17 ± 3.42a 0.44 ± 0.09a AF-WB 80.06 ±2.48c 69.50 ± 1.05a 1.28 ± 0.09d 56.24 ± 0.74a 0.42 ± 0.04a AF-MW 94.76± 1.96a 53.49 ± 0.86b 2.12 ± 0.08c 45.60 ± 2.24b 0.55 ± 0.02a Control62.00 ± 9.08d 18.18 ± 1.47d — 11.18 ± 2.31c — *Values are mean ±standard deviation of triplicate determinations. Different letters (a-d)indicate significant differences (p < 0.05) between treatments (rows).A-WB: chia seed protein hydrolyzed by alcalase enzyme using water bathheating method. A-MW: chia seed protein hydrolyzed by alcalase enzymeusing microwave-assisted hydrolysis. AF-WB: chia seed protein hydrolyzedsequentially by alcalase and flavourzyme enzymes using water bathheating method. AF-MW: chia seed protein hydrolyzed sequentially byalcalase and flavourzyme enzymes using microwave-assisted hydrolysis.Control: unhydrolyzed chia seed protein, also known as chia seed flour.

TABLE 2 Correlation analysis between antioxidant assays and cellularantioxidant analysis. Antioxidant Pearson Correlation assaysCoefficients for CAA (r) Probability MIC 0.8626 0.059 ABTS 0.881920.047* DPPH 0.88887 0.043* ORAC 0.64662 0.023* *significant correlation(p < 0.05) was established.

TABLE 3 Total Amino acid content (g/100 g) for chia seed protein andCSPH. Amino Acid Control A-WB A-MW AF-WB AF-MW Gly 5.128 4.278 4.4654.231 4.354 Ala 5.405 5.196 4.994 4.972 4.940 Pro 3.573 3.518 3.5333.477 3.708 Val 4.358 4.462 4.272 4.563 5.021 Ile 2.544 3.096 3.0033.114 3.474 Leu 5.328 5.685 5.550 5.532 5.985 Met 4.629 3.604 3.9863.970 3.759 Phe 3.267 3.219 3.408 3.397 3.850 His 1.417 1.358 1.3621.332 1.358 Thr 3.964 3.608 3.502 3.520 3.567 Cys-Cys 3.046 1.536 1.8431.797 1.804 Tyr 0.069 0.013 0.017 0.009 0.007 Glu 29.001 30.857 30.67530.552 29.181 Arg 7.309 7.484 7.985 8.125 8.316 Ser 4.913 5.447 5.5455.556 5.551 Asp 10.064 12.158 11.710 11.418 10.808 Lys 5.984 4.479 4.1514.436 4.317 SCAA* 7.675 5.140 5.829 5.767 5.564 AAA 4.754 4.591 4.7884.738 5.215 PCAA 14.710 13.322 13.498 13.893 13.991 BCAA 12.230 13.24312.825 13.208 14.480 *SCAA = sulfur containing amino acids (Met + Cys).AAA = Aromatic amino acids. (Phe, His and Tyr). PCAA = positivelycharged aminoacids (Arg, His and Lys). BCAA = Branched containing aminoacids (Leu, Ile and Val). Sample codes descriptions are provided inTable 1.

Enzymatic Hydrolysis

Table 1 shows the degree of hydrolysis obtained from the differenttreatments. The highest degree of hydrolysis (46.81±0.19 and40.68±0.77%) was obtained by sequential hydrolysis with alcalase andflavourzyme (AF-WB) and (AF-MW), respectively. CS protein is difficultto hydrolyze due to its high content of globulin fractions, whichcontain several sulfur amino acids directly involved in maintaining thetertiary and quaternary structure of the protein (Sandoval-Oliveros &Paredes-López, 2013). This conformation limits the access of the enzymeto cleavage sites that are located within the protein fraction. This wasconfirmed by the amino acid analysis were the content ofsulfur-containing amino acids (Cys+Met) (Table 3) make up to 7% of thetotal amino acid composition of chia seed flour (control); a similarvalue of 6% was reported by Sandoval-Oliveros and Paredes-López (2013).The higher hydrolysis in the AF-MW treatment can be attributed to theefficient separation of mucilage and the use of microwave energy.

Singh, Orsat, and Raghavan (2013) evaluated the effect ofelectromagnetic fields in protein structure and conformational changes,showing how magnetic forces pull dielectric charges in the proteinbackbone causing protein unfolding or re-orientation (Singh et al.,2013). The protein unfolding exposes active sites that allow theproteases to carry out digestion in a fast and efficient way. Inaddition, separation of mucilage played an important role in theeffectiveness of the hydrolysis. The minimum presence of thispolysaccharide can block the enzyme-substrate interaction by a processcalled enzyme immobilization, resulting in low proteolysis. A studyconducted by Monroy-Torres, Mancilla-Escobar, Gallaga-Solórzano, andSantiago-García (2008) showed that CS flour has a low proteindigestibility (79.28%) which is attributed to the presence of mucilage.In general, the utilization of an efficient mucilage separation methodand microwave energy decreased the time required to generate higherhydrolysis (DH) and consequently produce lower molecular weightpeptides.

Antioxidant Activity

The antioxidant properties of the peptides are shown in (Table 1).Overall, the trend for radical scavenging activity of the peptidesshowed an increase with increasing extent of hydrolysis (DH). AF-MWprotein hydrolysates showed overall significant higher (p<0.05)antioxidant properties for MIC, DPPH, ABTS, and CAA. Orona-Tamayo,Valverde, Nieto-Rendón, and Paredes-López (2015) identified that CSprotein between 20 and 33 kDa corresponds to the globulin proteinfraction. Globulin fractions, specifically Globulin 11 s, areresponsible for the high antioxidant activities in plant-based materials(Delgado et al., 2016). In this study, the same band was observed in themolecular weight distribution corresponding to AF-MW (FIG. 3); it issuspected that the combination of sequential hydrolysis and microwaveenergy caused a partial extraction of globulin fractions. Also, thepresence of small peptides in all CSPH treatments displayed strongmolecular weight bands below 25 kDa (FIG. 3), the presence of lowmolecular weight peptides can be responsible for the high antioxidantactivity of CSPH treatments when compare to the unhydrolyzed chia seedprotein (control).

For ABTS, the results for AF-MW are 70-fold higher than those previouslyreported by Segura-Campos, Salazar-Vega, Chel-Guerrero, andBetancur-Ancona (2013), where the protein hydrolysates were producedusing sequential hydrolysis and water bath. ABTS and DPPH assaysevaluated antioxidant activity by a specific mechanism called scavengingof free radicals. Studies have shown that ability is enhanced dependingon factors such as amino acid composition, protein sequences andstructural properties (Sarmadi & Ismail, 2010). AF-MW treatment allowedfor the release of a specific group of globulin fractions (G3, G4 andG5), which compared to unhydrolyzed CS protein, showed that the globulinfraction has a higher concentration of Phe, Tyr and His(Sandoval-Oliveros & Paredes-López, 2013). These results lined up withthe ones obtained in this study, were the content of aromatic aminoacids (His and Phe) are higher in AF-MW compared to unhydrolyzed CSprotein (Table 3). It is hypothesized that protein rotation andunfolding caused by MW and the release of encrypted peptides cause bysequential hydrolysis, enhanced the exposure of these aromatic aminoacids responsible of a high donation of protons (Sarmadi & Ismail,2010). The proton donations from aromatic amino acids to molecules withelectron deficiency, improves the scavenging activity stabilizingreactive molecules (Sarmadi & Ismail, 2010). In contrast, the metal ionchelating (MIC) capacity is generally attributed to peptides containingsulfhydryl amino acids such as Cys and Met, which can bind heavy metalsand reduce the pro-oxidant activity of some metals. The content of Cysand Met amino acids (Table 3) was higher in the control than in othertreatments. It is hypothesized that microwave-assisted hydrolysisreleased encrypted sulfur peptides, making them more bioavailable tointeract with free metals, therefore enhancing the MIC capacity.

ORAC is a common antioxidant assay based on the quantification of thefluoresce emitted by a probe, in this case a protein called fluorescein.Fluorescein is exposed to an oxidative environment by AAPH that causesdegradation of the protein, and, consequently, a loss in fluorescence.This assay measures the hydrophilic antioxidant capacity against certainperoxyl radicals (Aruoma, 2003). The highest ORAC values (p<0.05) wereseen in the microwave treatments A-MW and AF-WB at, 1535.81±99.16 and1482.48±114.63 μmol TE/mg of protein, respectively. Exposure duringhydrolysis of encrypted nonpolar residues such as Gly, Ala, Ile, Trp,Tyr and Met is speculated, this in turn can cause an increase inhydrophobic interactions of peptides with oxidizing agents. Theseresults showed a higher ORAC value compared to other digested materialssuch as cowpea protein (783.8 μmol/g of protein) and salmon (1541 μmol/gof protein) (Marques et al., 2015).

The cellular antioxidant activity (CAA) assay indirectly evaluates thepermeability of a compound through the cell bilayer while estimating theantioxidant capacity. In this method, a fluorescent probe DCFH-DA isintroduced into the Caco-2 cells, and by the action of esterase isturned into a more polar form of DCFH which emits fluorescence underphysiological conditions (Wan et al., 2015). In order to protect DCFHfrom oxidation caused by AAPH solution, the CSPH peptides must permeateand/or bind to the cell membrane and function a protective barrieragainst free radicals, thus preventing molecular damage of intracellularorganelles and proteins. If the peptides have a good permeability and ahigh enough antioxidant capacity, they will protect the fluorescentprobe from turning into dichloro-dihydro-fluorescein (DCF) andconsequently avoiding a decrease in light emission through time (Wan etal., 2015). The highest CAA value was observed for the microwavetreatments AF-MW and A-MW (94.76±1.96 and 93.13±1.07%, respectively).Wolfe and Liu (2007) found that the hydrophobicity of the compounds wasan important criterion to determine the antioxidant effectiveness incell culture. This is not the only criteria since structuralconformation also plays a crucial role in the quality and effectivenessof the antioxidant activity of these compounds (Wolfe & Liu, 2007).Conradi, Hilgers, Ho, and Burton (1991) found that the permeability ofpeptide chains was significantly correlated with the total number ofhydrogen bonds a peptide can possibly form with water since the majorimpediment for passive absorption is the energy required to break thepeptide-water hydrogen bonds. It is hypothesized that smaller peptidesthat have a weaker hydrogen bonding capacity and a higher concentrationof antioxidant amino acids on the microwave samples (A-MW and AF-MW),are responsible for the increased cellular antioxidant activity observedin this study.

Correlation of Antioxidant Assays to CAA

A correlation analysis was carried out determining the significance ofsome antioxidant assays to predict cellular antioxidant activity. Theanalysis presented a significant correlation (p<0.05, r=0.888) of DPPHwith respect to CAA followed by ABTS (Table 2). No significantcorrelation was found for MIC and CAA. The correlation values betweenantioxidant assays and CAA was not found in literature for most of theconventional antioxidant methods. Wolfe et al. (2008) established acorrelation of ORAC to CAA finding that this assay positively correlateswith a Pearson correlation coefficient of (r=0.803) and a significance(p<0.05). Their results differ with the results obtained in this study,were ORAC values showed lower correlation (r=0.646, p<0.05). We canconclude that DPPH and ABTS are the most useful screening methods forantioxidant evaluation of compounds since the chemical conditions ofthese assays allow the effective prediction of in vitro antioxidantactivity in cellular environments.

Antidiabetic Properties

The antidiabetic capacity assay measures the peptides' ability toinhibit human DPP-IV. The DPP-IV activity improved in all hydrolyzedtreatments compared to the control (Table 1). The highest (p<0.05)DPP-IV inhibition was observed in AF-WB (69.50±1.05%) and the lowest wasin the control (18.18±1.47%). Matsui, Oki, and Osajima (1999) studiedthe DPP-IV inhibition activity of sardine muscle hydrolysates andattributed the inhibition capacity to di- and tetra-peptides that matchthe structure of the substrate of the DPP-IV enzyme. In another study,Nongonierma, Le Maux, Dubrulle, Barre, and FitzGerald (2015) evaluatedthe DPP-IV inhibitory activity of the protein hydrolysates of quinoa, asimilar pseudo-cereal. The IC50 value of quinoa protein hydrolysates wasof 0.88±0.05 mg/mL using a porcine DPP-IV enzyme (0.0025 U/mL).Velarde-Salcedo et al. (2013) evaluated the DPP-IV inhibition ofamaranth tryptic digests using porcine DPP-IV (0.0025 U/mL) and foundIC50 value ranging between 1.2 and 2 mg/mL. To the best of authors'knowledge, no previous report of DPP-IV inhibition activity by CSPHusing human DPP-IV (2.25 U/mL) has been reported. In this study AF-WBCSPH had an IC50 of 1.28 mg/mL, showing the bioactive potential forCSPH, compared to other pseudo-cereals. Lacroix and Li-Chan (2015)compared the susceptibility of porcine and human DPP-IV to inhibitionand found that porcine DPP-IV is generally inhibited with greaterefficacy by protein derived peptides than human DPP-IV. Generally, theDPP-IV inhibition is enhanced with the presence of lower molecularpeptides, probably matching the structure of the incretin hormones (GIPand GLP-1). The incorporation of flavourzyme might have influenced thedevelopment of tetra- and di-peptides that match the DPP-IV active site.A similar result was obtained for protein hydrolysates derived fromAtlantic salmon (salmon salar) skin gelatin, were the highest DPP-IVinhibition was achieved using flavourzyme (Li-Chan, Hunag, Jao, Ho, &Hsu, 2012).

Anti-Hypertensive Activity

In this study, most of the CSPH treatments had similar ACE inhibitioncapacity except for AF-MW and the control which were significantly(p<0.05) lower (Table 1). Other studies have looked at the bioactivityof CSPH incorporated into foods. A study conducted by Segura-Campos etal., 2013, showed an improvement in IC50 value of the ACE-inhibitorycapacity of foods by incorporating 5.0 mg/g of CSPH into carrot cream,causing a decrease from 27.67 μg protein/mL to 1.71 μg protein/ml(Martínez-Hernández, Orona-Tamayo, Valverde-González, & Paredes-López,2017). In our study, the IC50 values of CSPH was around 0.40 mg/mL. Inthe case of the lower inhibitory activity of AF-MW, it might be relatedto the formation of different structural peptides with a lower bindingcapacity to the ACE active site. Additional Analytical studies areneeded to understand the difference with peptide conformation in CSPH.It is well known that the increase in DH and lower molecular-weightpeptides improve the capacity of inhibiting ACE enzymes. Segura-Campos,Peralta-González, Chel-Guerrero, and Betancur-Ancona (2013) evaluatedthe ACE inhibitory capacity of CSPH (51.64% DH) and obtained 53.8-69.3%inhibition for purified fractions. In this study, the CSPH peptidesdisplayed a 57% inhibition with a 46% DH. Nevertheless, an influence ofmicrowave energy in peptide conformation is not clear for ACEinhibition. Ketnawa, Suwal, Huang, and Liceaga (2019) evaluated the ACEinhibition of microwave-assisted hydrolyzed peptides of rainbow trout(Oncorhynchus mykiss) and showed that peptides delivered frommicrowave-assisted hydrolysis had the highest inhibition at 93.5±0.24%.

Functional Properties

Solubility. The solubility of CSPH showed a dependency of the pH in alltreatments, with solubility increasing with increasing pH (FIG. 2A).This pattern is similar to what is reported for many plant proteins, andis related to the low isoelectric point (pI) of CS protein. When the pHof the solution is above or below the pI of a protein the solubility isenhanced because the electrostatic repulsion between molecules isgreater than the hydrophobic interactions (Zayas, 2012). CS proteinextraction is more efficient at alkaline pH, reaching its maximumsolubility at pH 12 (Timilsena, Adhikari, Barrow, & Adhikari, 2016). Aprevious study by Timilsena et al. (2016) evaluated the solubility of CSprotein isolate, reporting 10% solubility at pH 3. A similarly low valuewas obtained in this study for the solubility of the control (16%) (FIG.2A). AF-MW treatment showed the highest solubility at pH 3 (68.32%) andpH 5 (77.17%) when compared to other treatments (FIG. 2A). This can beattributed to the high degree of hydrolysis of this treatment (40.68%),where the presence of small peptides increases the exposure of polar andionizable groups, consequently increasing their solubility (Nguyen etal., 2017). In addition, enzymatic hydrolysis by alcalase andflavourzyme can increase the number of smaller hydrophilic polypeptides(Zhao et al., 2012). At neutral and alkaline pH, A-MW treatment showedthe highest solubility (p<0.05), suggesting that a medium DH (mediumsize peptides) could improve the solubilization of protein in neutraland alkaline solvents. The application of microwave-assisted hydrolysiscan be correlated with an increase production of small peptides andconsequently with an increased solubility (Uluko et al., 2013). Overall,hydrolyzing the CS flour improved the solubility of the proteinhydrolysates, compared to the unhydrolyzed control at all pH rangevalues.

Emulsion and Foaming Capacity

Hydrolysis of CS flour increased its emulsifying capacity (at 0 min)compared to the control (FIG. 2B). No difference (p<0.05) was found forthe emulsifying capacities between A-WB, A-MW, and AF-MW. The overallimprovement of these treatments in relationship with the control(unhydrolyzed protein) may be due to the enzymatic digestion byalcalase. Other researcher have reported similar observations. Forexample, Klost and Drusch (2019) hydrolyzed pea protein using enzymatichydrolysis, and found that the oil-droplet-size in hydrolyzed proteinswas smaller increasing the emulsion capacity of the unhydrolyzedprotein, they attributed this to a vast exposure of hydrophobic residuesand the presence of electrostatic repulsion between droplets. Inaddition, a possible effect of microwave irradiation is thought to be aresult of protein denaturation. Villanueva, Harasym, Muñoz, and Ronda(2019) evaluated the effect of microwave energy in the viscoelasticcharacteristics of rice flour, where microwave treatments caused andoverall increase in the network formation, which was attributed to thedenaturation of the protein. Similarly, Zhang et al. (2019) evaluatedthe effect of heat treatments in camelia (C. oleifera) protein cake andfound that protein denaturation caused a modification in the secondarystructure of the protein. In contrast, AF-WB had lower emulsifyingcapacity (p<0.05) followed by the control. The decreased emulsifyingcapacity in this treatment could be correlated to its high degree ofhydrolysis, meaning there is a higher content of low molecular weightpeptides. In a study conducted using quinoa seed protein hydrolysates,results showed how smaller peptides are unable to form stable filmsaround oil droplets, which result in the emulsion collapsing within time(Aluko & Monu, 2003). Differences (p<0.05) were observed when evaluatingthe emulsifying stability over 30, 60 and 90 min, where A-MW showed thebest stability at 30 and 60 min, followed by the AF-MW (FIG. 2B).However, both alcalase treatments (A-MW and A-WB) had similar emulsionstability at 90 min. A study conducted by van der Ven, Gruppen, de Bont,and Voragen (2001) showed that whey and casein protein hydrolysates havebetter emulsifying capacities compared to intact casein and wheyprotein. Authors attributed this to a more uniform distribution ofemulsion droplets size originated from partially hydrolyzed peptidescompared to intact protein were the droplets are bigger and collapsefaster (van der Ven et al., 2001).

Foaming. A-MW showed the highest foaming capacity (75%) (at 0 min),followed by A-WB (66.5%) (FIG. 2C). This result agrees with the highestsolubility at pH 7 observed in A-MW, indicating that these peptides aremore evenly distributed through the aqueous interface and consequentlyimprove the way they entrap air molecules. Similarly, Nguyen et al.(2017) developed protein hydrolysates from rainbow trout frames usingmicrowave-assisted hydrolysis, proving that lower molecular weightpeptides derived by microwave energy diffuse faster into the air-waterinterface. CSPH showed a great potential in foaming capacity whencompared to other protein sources such as whey protein hydrolysates andrice protein hydrolysates with 4 and 6% foaming capacity, respectively(Amagliani, O'Regan, Schmitt, Kelly, & O'Mahony, 2019). Olivos-Lugo,Valdivia-López, and Tecante (2010) evaluated the foaming capacity of CSprotein isolate and reported 70% foaming capacity. The purification andisolation of the protein is suspected to be responsible of the highvalues reported for the CS protein isolate. Evaluation of foam stabilityat 30, 60 and 90 min, showed that A-WB resulted in overall higherfoaming stability. The decrease in foaming capacity of A-MW compared toA-WB can be attributed to the presence of smaller peptides caused bymicrowave treatment; smaller peptides are known to have poor stabilitythrough time. Nevertheless, A-WB and A-WW have lower DHs amongst allCSPH (Table 1) meaning that medium size peptides might be present. Baltiet al. (2010) evaluated the influence of DH on foaming properties ofcuttlefish and found that foaming capacity decreased slightly as theprotein hydrolysis increased. They attributed this to the loss ofcohesiveness that is achieved with high molecular-weight peptides andpartially hydrolyzed protein.

Anti-Aging Bioactive Properties

The inhibition properties of bioactive peptides towards the main enzymesassociated with skin aging can indicate their potential role in theimprovement of skin health. Our results (FIG. 5) showed that the <3 kDapeptide fraction (at 1 mg/mL) exhibited inhibitory activity towards allaging-related enzymes. Overall, this peptide fraction showed <50% ofinhibition on collagenase and hyaluronidase enzymes, while itshowed >50% of inhibition on tyrosinase and elastase enzymes.Accordingly, the IC₅₀ values showed a similar trend, obtaining the bestinhibitory value (P<0.05) on elastase (0.43 mg/mL), followed bytyrosinase (0.66 mg/mL), hyaluronidase (1.28 mg/mL), and collagenase(1.41 mg/mL).

Mode of Enzyme Inhibition

In order to investigate the inhibition pattern of chia peptides on theaging-related enzymes, each enzyme was assayed with differentconcentrations of substrate with and without inhibitor; such modes ofinhibition are illustrated in Lineweaver-Burk plots (FIGS. 6A-6D). Chiaseed peptides showed a mixed-type inhibition pattern towards elastase(FIG. 6A) and hyaluronidase (FIG. 6C) characterized by Km and Vmaxchanges at different concentrations. Chia peptides showed anon-competitive inhibition pattern towards collagenase (FIG. 6B) andtyrosinase (FIG. 6D), as seen by a decrease in Vmax and with constantKm.

Peptides Associated with Enzyme Inhibition in the Most Active <3 kDaPeptide Fraction

In order to identify the peptides associated with each enzymeinhibition, the <3 kDa peptide fraction was fractionated using sizeexclusion chromatography (SEC). The peptide chromatographic profile wasdivided into 5 fractions (FIG. 7) and collected for further evaluation.Overall, all SEC peptide fractions showed inhibitory activity towardsskin aging-related enzymes, except for fraction V, which did not exhibitinhibitory activity towards collagenase (Table 5). Fractions II (F-II)and IV (F-IV) showed the highest inhibitory activity for most of theenzymes. Particularly, F-II exhibited the highest (P<0.05) collagenase(38.01%) and elastase (55.61%) inhibitory activities; followed by F-IVwith elastase (42.92%) and tyrosinase (61.81%) inhibitory activities.Interestingly, some fractions showed higher enzyme inhibitory activitycompared to their counterpart <3 kDa peptide fraction. For instance, theF-II showed higher collagenase (38.01%) or elastase (55.61%) inhibitoryactivity compared to 28.90 and 65.32%, respectively, in the <3 kDapeptide fraction.

In Silico Analysis of Elastase Peptide Interactions

Based on our results, the F-II was further selected to identify thepeptide sequences involved in bioactivity and was subjected to dockingmodeling to observe the enzyme-peptide interactions. A total of sevenpeptides were identified in F-II, APHWYTN (SEQ ID NO: 1), DQNPRSF (SEQID NO: 2), GDAHWAY (SEQ ID NO: 3), GDAHWTY (SEQ ID NO: 4), GDAHWVY (SEQID NO: 5), GFEWITF (SEQ ID NO: 6), and KKLKRVYV (SEQ ID NO: 7). Thesepeptides were used in docking analysis to generate predicted models ofinteraction between elastase and each peptide sequence (FIG. 8). Thesemodels predicted that in the interaction of elastase and the peptidesequence, between 19 to 29 pair protein-peptide residues participated(Table 6). Moreover, five peptide sequences (APHWYTN (SEQ ID NO: 1),DQNPRSF (SEQ ID NO: 2), GDAHWAY (SEQ ID NO: 3), GDAHWTY (SEQ ID NO: 4),and KKLKRVYV (SEQ ID NO: 7)) exhibited hydrogen bonding betweenprotein-peptide residues. In addition, it is important to note thatthree peptide sequences share structure homology sequence (GDAHW (SEQ IDNO: 8)). In addition, we calculated the frequency of pair interactionsbetween elastase and all seven peptides sequences obtained from F-II(FIG. 9). This graph allows us to characterize the binding sitesdetected in elastase and graphically analyze if they are the same forthe different peptide sequences. We observed that specific elastaseamino acid regions participated repeatedly in the interactions with thepeptide sequences. These main regions were 38 W-49 Q, 88 I-129 I, 168C-188 R, and 204 N-230 R. Particularly, the second region (88 I-129 I)showed interesting features by containing the elastase amino acid (89 V)with seven interactions, the highest amongst all sites. In addition, itshowed a continuous sequence of interaction between the positions121-129. In contrast, these interaction regions are close to or at leastin vicinity to the active site of elastase, consisting of the aminoacids 71 H, 119 D, and 214 S.

Summary and Conclusions

It is known that chia seed is a complex food composed of highpolysaccharide, protein, and oil content. The implementation ofultrasonication and vacuum-filtration, successfully improved theseparation of mucilage from chia seeds (7.8% yield) compared to previousextraction methods using sieves and conventional ovens. The efficientseparation of mucilage followed by lipid extraction using a coldscrew-press facilitated the separation of protein-rich chia seed flour.Microwave-assisted hydrolysis with alcalase and flavourzyme improvedbioactivity and functionality of the CSPH in a shorter amount of timecompared to conventional hydrolysis methods. CSPH from the sequentialhydrolysis with microwave treatment showed overall superior in vitroantioxidant activity. Cellular antioxidant activity showed the potentialantioxidant activity of these peptides for future use in vivo models,with AF-MW having the highest cellular antioxidant activity (94.8%). Apositive correlation between antioxidant assays and cellular antioxidantactivity was established, showing that ABTS (r=0.882) and DPPH (r=0.889)were the most efficient assays in predicting cellular antioxidantactivity of CSPH. DPP-IV inhibition increased as a function of DH,suggesting that higher hydrolysis will result in improved inhibitions.DPP-IV inhibition was the highest for AF-WB (69.5%), followed by AF-MW(53.5%), suggesting the benefit of using sequential enzyme hydrolysis.For ACE inhibition, all CSPH showed improved inhibition activitycompared to the control; however, no apparent influence was foundregarding type of hydrolysis (water bath versus microwave) in theirinhibition capacity, suggesting the structural conformation responsibleof this bioactivity remains unchanged independently of the treatmentapplied. Lastly, further studies are required regarding the structuralconformation of these peptides to fully understand their inhibitionmechanism.

Applying different technologies such as those used in this study,facilitated the separation of the chia seed components. In addition,hydrolyzing the chia seed protein allowed for development of proteinhydrolysates with functional and bioactive properties, which will allowfor their applicability in food science or in the pharmaceutical sector.

To best of our knowledge, this is the first study that reports theaging-related enzyme (i.e., elastase, tyrosinase, hyaluronidase, andcollagenase) inhibitory properties of chia seed peptides. Overall, ourresults showed that the <3 kDa peptide fraction exhibited inhibitoryactivity towards all enzymes tested, with mixed-type (e.g. elastase andhyaluronidase) or non-competitive (e.g. collagenase and tyrosinase)inhibition patterns. Furthermore, the peptide fraction F-II showed thehighest potential for elastase inhibition. Based on in silico peptidedocking analysis, chia peptide sequences identified in F-II (APHWYTN(SEQ ID NO: 1), DQNPRSF (SEQ ID NO: 2), GDAHWAY (SEQ ID NO: 3), GDAHWTY(SEQ ID NO: 4), GDAHWVY (SEQ ID NO: 5), GFEWITF (SEQ ID NO: 6), andKKLKRVYV (SEQ ID NO: 7)) could bind to either the enzyme alone and/orthe enzyme-substrate complex. The inhibition occurs due to 19-29elastase-peptide pair interactions, possibly with the active site orother recognition motifs located on the enzyme surface. Based on thedocking analysis, our results suggest that chia seed peptides possessamino acids that participate during the enzymatic inhibitory process byestablishing different protein-peptide pair interactions, includinghydrogen bonding. In addition, three peptide sequences shared structurehomology sequence (GDAHW (SEQ ID NO: 8)), which could play a predominantrole in their inhibitory activity. The data suggests that these peptidesequences may contribute in the improvement of skin health by offeringprotection against aging-related enzymes such as elastase, by avoidingthe degradation of the protein matrix of the skin (FIG. 10).

Exemplified Process:

Step 1. Chia Seed Mucilage Extraction

To extract the chia seed mucilage (FIG. 1), seeds are hydrated indistilled water (1:20 ratio by weight) for 24 hours, under refrigeratedconditions. Preliminary studies (Table 4) helped develop an ultrasoundtreatment that offered successful mucilage separation. Hydrated seedswere pre-heated to 55±2° C., followed by sonication at a 75% power inputusing an ultrasonic cell disruptor QSonica Q500 Sonicator (QSonica LLC,Newton, Conn., USA). During sonication, the temperature increased to60±4° C. due to molecular friction. This temperature was maintainedconstant using double walled beaker connected to an immersion circulatorcontrol Lauda E100 water bath (Lauda-Königshofen, Germany). Seeds wereseparated from mucilage using vacuum-assisted filtration system.

Mucilage-free chia seeds were dried using a tray dryer (ExcaliburDehydrator 3926TCDB, Sacramento, Calif.) held at 40° C. for 12 h. Theweight of the seeds was measured to calculate mucilage extraction yieldby weight difference [Eq.12]. Finally, the mucilage solution was frozenat −85° C. and lyophilized (Labconco FreeZone 2.5 Plus, Kansas City).

Dried mucilage powder was stored at 4±2° C. until used.

$\begin{matrix}{{\% \mspace{14mu} {mucilage}\mspace{14mu} {yield}} = {\frac{\begin{pmatrix}{{{Weight}\mspace{14mu} {of}\mspace{14mu} {dry}\mspace{14mu} {chia}\mspace{14mu} {seeds}} -} \\{{Weight}\mspace{14mu} {of}\mspace{14mu} {dry}\mspace{14mu} {chia}\mspace{14mu} {seeds}\mspace{14mu} {without}\mspace{14mu} {mucilage}}\end{pmatrix}}{{Weight}\mspace{14mu} {of}\mspace{14mu} {dry}\mspace{14mu} {chia}\mspace{14mu} {seeds}} \times 100}} & \left\lbrack {{Eq}.\mspace{11mu} 12} \right\rbrack\end{matrix}$

TABLE 4 Mucilage extraction from chia seeds under different temperatureand separation conditions. Temperature Yield of mucilage (° C.)Separation method extraction (%) 55 Vacuum-filtration 7.66 ± 0.23 55Centrifugation 6.23 ± 0.56 40 Vacuum-filtration 4.72 ± 0.17 40Centrifugation 3.66 ± 0.17 25 Vacuum-filtration 3.66 ± 0.17 25Centrifugation ±0.17

Step 2. Chia Seed Oil Extraction

Dried, mucilage-free chia seeds were defatted using a mechanical oilextraction method with a Beamnova Automatic Oil Press Machine(Commercial 304 Stainless Steel Expeller, Guangzhou, China). Seeds werepressed using a stainless-steel endless screw where the temperature ofthe oil extracted from the seeds was 37±2° C. Percentage of oilextraction was calculated by weight difference [Eq.13]. Hydrogen gasadded to the oil held in plastic containers and stored at 4° C. Thedefatted chia seed flour without mucilage was subject to enzymatichydrolysis as a way to improve protein extraction, functional(solubility, emulsion and foaming), and bioactive (antidiabetic,antioxidant, antihypertensive) properties.

$\begin{matrix}{{{oil}\mspace{14mu} {yield}\mspace{14mu} \%} = {100 \times \frac{\begin{pmatrix}{{{Weight}\mspace{14mu} {of}\mspace{14mu} {chia}\mspace{14mu} {seeds}} -} \\{{Weight}\mspace{14mu} {of}\mspace{14mu} {defatted}\mspace{14mu} {chia}\mspace{14mu} {seeds}\mspace{14mu} {flour}}\end{pmatrix}}{{Weight}\mspace{14mu} {of}\mspace{14mu} {chia}\mspace{14mu} {seeds}}}} & \left\lbrack {{Eq}.\mspace{11mu} 13} \right\rbrack\end{matrix}$

The oil extraction yield obtained by cold press was 28.24±0.06 g ofoil/100 g of chia seeds (without mucilage). The oil content in chiaseeds varies between 30-33% (Sandoval-Oliveros & Paredes-López, 2013).Cold pressing of chia seeds without mucilage allows to remove about95.81±0.12% of the total fat of the seed. Conversely, CS with mucilagelead to an extraction of only 62.90±0.77%. This is mainly attributed tothe high oil holding capacity of chia seed mucilage (Darwish, Khalifa, &El Sohaimy, 2018).

TABLE 5 Inhibitory activity of skin aging-related enzymes by sizeexclusion chromatography (SEC) peptide fractions obtained from <3 kDachia seed protein hydrolysates. SEC fraction I II III IV V Skin-agingenzyme % inhibition Collagenase  10.50 ± 3.88 a* 38.01 ± 3.44 c 20.13 ±5.68 b 22.83 ± 3.61 b N.D. Elastase 23.85 ± 3.35 b 55.61 ± 3.91 d 12.79± 2.76 a 42.92 ± 2.38 c 15.98 ± 1.32 a Hyaluronidase 25.20 ± 3.81 b 7.43 ± 1.29 a 33.39 ± 5.96 b 16.18 ± 2.35 a 48.61 ± 5.19 c Tyrosinase33.93 ± 1.82 c 24.33 ± 2.68 b  7.08 ± 1.78 a 61.81 ± 3.50 d 21.57 ± 4.36b *Different lowercase letters indicate statistical differences amongSEC fractions (column) for the same enzyme. N.D. Not detected. Valuesrepresent mean ± standard deviation of triplicate determinations.

Step 3. Chia Seed Protein Hydrolysates (CSPH)

Chia flour was diluted in distilled water to obtain 22.5 mg protein/mLand homogenized using a Sorvall Omni Mixer homogenizer with amacro-attachment assembly (Norwalk, Conn., U.S.A). The pH was adjustedto 8.0 using 2 M NaOH, which is the optimal condition for alcalaseactivity. Proteins were enzymatically hydrolyzed using single enzymealcalase (A) or a sequential digestion with alcalase+flavourzyme (AF).Proteolysis occurred using conventional/water bath (WB) ormicrowave-assisted (MW) hydrolysis using a microwave acceleratedreaction system (MDS, MARS-Xpress/230/60, CEM Corporation, USA).Treatments were denoted as conventional alcalase hydrolysis using awater bath (A-WB) and alcalase microwave-assisted hydrolysis (A-MW).Sequentially (AF) hydrolyzed treatments were coded as AF-WB (water bathhydrolysis) and AF-MW (microwave-assisted hydrolysis). Finally, thecontrol (C) was non-hydrolyzed CS protein. Samples A-WB and A-MW werehydrolyzed for 1 hour with 3% (w/w) Alcalase. For sequential hydrolysisdifferent times were used, due to the high efficiency ofmicrowave-assisted hydrolysis the time was cut down by half to obtainsimilar extent of hydrolysis. AF-MW, the reaction was initiated with 2%(w/w) of Alcalase for 45 min followed by addition of 2% (w/w) ofFlavourzyme for an additional 45 min. For AF-WB, the reaction wasdeveloped using 2% (w/w) of Alcalase for 90 min followed by 2% (w/w) ofFlavourzyme for another 90 min. Hydrolysis was terminated by heating to95±3° C. for 15 min. The protein slurry mixture was centrifuged at17,636×g and the supernatant was recovered as a source of protein. Thesupernatant was frozen at −85° C. and lyophilized (Labconco FreeZone 2.5Plus, Kansas City). A detailed process for the preparation of the Chiaseed products (mucilage, oil, and protein) ingredients is summarized inFIG. 4.

TABLE 6 Pairs of elastase and peptide amino acids that participated inthe interaction from the protein-peptide predicted model. APHWYTNDQNPRSF GDAHWAY GDAHWTY No. Enzyme Peptide Enzyme Peptide Enzyme PeptideEnzyme Peptide pair residue residue residue residue residue residueresidue residue 1 N 245 P 2 N 245 Q 2 K 177 A 6 R 230 T 6 2 V 203 N 7 N239 N 3 T 175 A 3 N   178 W   5 3 R 125 Y 5 N 178 R 5 S 174 W 5 K 177 W5 4 L   123* Y 5 D 164 F 7 Y 171 H 4 I 167 D 2 5 V 122 W 4 T 128 S 6 D97 D 2 T 128 T 6 6 L 114 Y 5 G 127 R 5 T 96 A 3 A 126 T 6 7 I 47 Y 5 Y93 P 4 V 90 A 3 Y 93 H 4 8 I 47 A 1 Y 93 D 1 V 89 G 1 N 204 Y 7 9 Q 206Y 5 N 245 N 3 S 217 G 1 N 178 D 2 10 R 125 T 6 S 244 N 3 T 175 H 4 S 170D 2 11 P 124 Y 5 A 233 P 4 S 174 A 6 T 128 Y 7 12 V 122 Y 5 Y 165 F 7 G173 H 4 A 126 Y 7 13 G 121 Y 5 T 128 F 7 D 97 A 3 W 94 H 4 14 W 51 P 2 G  127 S   6 T 96 H 4 R 230 W 5 15 I 47 P 2 R 125 P 4 H 91 W 5 N 178 H 416 V 209 Y 5 Y 93 Q 2 V 89 D 2 K 177 D 2 17 R 125 N 7 S 244 P 4 S 217 D2 I 129 W 5 18 P 124 T 6 Y 234 Q 2 K 177 W 5 T 128 W 5 19 L 123 W 4 K177 R 5 T 175 D 2 P 124 Y 7 20 V 122 H 3 I 129 F 7 S   174 H   4 21 L114 H 3 G 127 F 7 S 170 H 4 22 I 47 H 3 A 126 R 5 D 97 G 1 23 Y 93 N 3 T96 D 2 24 V 89 A 3 GDAHWVY GFEWITF KKLKRVYV No. Enzyme Peptide EnzymePeptide Enzyme Peptide pair residue residue residue residue residueresidue 1 N 245 D 2 R 217 F 2 R 223 V 8 2 V 209 H 4 C 191 F 2 T 222 Y 73 Y 207 G 1 D 98 W 4 G 187 Y 7 4 R 125 D 2 V 89 T 6 D 186 R 5 5 L 123 H4 V 89 E 3 C 168 V 6 6 N 115 H 4 I 88 W 4 Y 159 K 4 7 L 114 W 5 T 41 W 4G 19 K 1 8 Q 49 V 6 A 39 G 1 T 222 V 8 9 R 48 H 4 W 38 G 1 R 188 K 2 10I 47 H 4 R 217 E 3 D 186 Y 7 11 N 245 A 3 Q 192 F 2 C 168 Y 7 12 V 241 Y7 V 99 W 4 P 161 K 4 13 A 208 G 1 D 97 F 2 T 20 K 1 14 N 204 D 2 V 89 W4 R 223 Y 7 15 P 124 D 2 I 88 T 6 R 188 K 4 16 N 115 W 5 H 57 W 4 G 187K 4 17 L 114 V 6 A 39 F 2 D   186 K   4 18 Q 49 Y 7 W 38 W 4 C 168 R 519 R 48 W 5 S 217 F 2 Y 159 L 3 20 I 47 W 5 R 145 G 1 21 I 242 A 3 D 98F 2 22 A 208 H 4 V 89 I 5 23 N 204 H 4 I 88 F 7 24 P 124 H 4 V 59 F 7 25V 122 H 4 A 39 W 4 26 N 115 G 1 W 38 I 5 27 N 50 Y 7 28 R 48 Y 7 29 R 48A 3 *The bold, underlined pairs represent the amino acids thatparticipated in hydrogen bonding observed in the FIG. 8.

While this invention has been described as having an exemplary design,the present invention may be further modified within the spirit andscope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the invention using itsgeneral principles.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

While the inventions have been illustrated and described in detail inthe drawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly certain embodiments have been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected. It is intended that the scope of thepresent methods and apparatuses be defined by the following claims.However, it must be understood that this disclosure may be practicedotherwise than is specifically explained and illustrated withoutdeparting from its spirit or scope. It should be understood by thoseskilled in the art that various alternatives to the embodimentsdescribed herein may be employed in practicing the claims withoutdeparting from the spirit and scope as defined in the following claims.

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1. A process for manufacturing a plurality of products from chia seedscomprising the steps of: a. socking chia seeds in about 10 to 20 volumesof water for a period of time; b. sonicating soaked chia seeds using 75%power input for about 5 minutes at an elevated temperature to afford amixture; c. separating chia seeds from said mixture by vacuum filtrationto afford a solution, which is lyophilized to afford a mucilage productof chia seeds, a soluble fiber product; d. drying mucilage-removed chiaseeds; and e. extracting oil from dried chia seeds at an elevatedtemperature to afford an oil product and a flour of defatted chia seeds.2. The process according to claim 1 further comprising a step ofhydrolyzing the flour of chia seeds in an aqueous solution in presencean enzyme at a pH of about 6˜8.
 3. The process according to claim 2,wherein said hydrolyzing step is a microwave-assisted hydrolyzingprocess.
 4. The process according to claim 2, wherein said enzyme isalcalase optionally together with flavourzyme.
 5. The process accordingto claim 2, wherein said hydrolyzing step affords a solid product and asolution product after separation, wherein said solid product is aninsoluble fiber product of chia seed and said solution product is aprotein hydrolysate of chia seeds.
 6. The process according to claim 5,wherein said protein hydrolysate of chia seeds is further resolved intoa plurality of fractions comprising biologically active proteins andpeptides.
 7. The process according to claim 6, wherein said activepeptides have a sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or
 8. 8.The process according to claim 1, wherein said oil product of chia seedshas a yield of about 30% weight of starting chia seeds.
 9. The processaccording to claim 1, wherein said elevated temperature ranges fromabout 30° C. to about 70° C.
 10. A product manufactured according to theprocess of claim 1, wherein said product is an oil of chia seeds, amucilage of chia seeds, a soluble fiber product, or a soluble proteinhydrolysate.
 11. The product according to claim 10, wherein said solubleprotein hydrolysate comprises a peptide, a salt, a derivative or afragment thereof, having a sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,or
 8. 12. A plurality of products of chia seeds manufactured accordingto a process comprising the steps of: a. socking chia seeds in about10-20 volumes of water for a period of time; b. sonicating soaked chiaseeds using 75% power input for about 5 minutes at an elevatedtemperature to afford a mixture; c. separating chia seeds from saidmixture by vacuum filtration to afford a solution, which is lyophilizedto afford a mucilage product of chia seeds; d. drying mucilage-removedchia seeds; e. extracting oil from dried chia seeds at an elevatedtemperature to afford an oil product and a flour of defatted chia seeds;and f. hydrolyzing the flour of defatted chia seeds in an aqueoussolution in presence an enzyme.
 13. The products according to claim 12,wherein the step of hydrolyzing the flour of defatted chia seeds affordsa solid product and a solution product after separation, wherein saidsolid product is an insoluble fiber product of chia seed and saidsolution product is a protein hydrolysate of chia seeds.
 14. Theproducts according to claim 13, wherein said hydrolyzing step is amicrowave-assisted hydrolyzing process.
 15. The products according toclaim 13, wherein said enzyme is alcalase optionally together withflavourzyme.
 16. The products according to claim 13, wherein saidprotein hydrolysate of chia seeds is further resolved into a pluralityof fractions comprising active proteins and peptides.
 17. The productsaccording to claim 16, wherein said biologically active peptides have asequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or
 8. 18. The productaccording to claim 13, wherein said soluble protein hydrolysatecomprises a peptide, a salt, a derivative or a fragment thereof, havinga sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or
 8. 19. The productsaccording to claim 12, wherein said elevated temperature ranges fromabout 30° C. to about 70° C.
 20. The products according to claim 12,wherein said oil product of chia seeds has a yield of about 30% weightof starting chia seeds.